1. Charting the cellular interactome by proteome-wide cross-linking mass spectrometry  
Fan Liu
Utrecht University, The Netherlands
Netherlands Proteomics Center

2. Introduction of cross-linking mass spectrometry (XL-MS)
XL-MS workflow
Maximal distance constraints
Linker length
Sidechain
length
Distance constraint between C
11.4 Å
6.4 Å
24.2 Å
11.4 Å
DSS
Disuccinimidyl suberate

3. Introduction of cross-linking mass spectrometry (XL-MS)
XL-MS workflow

4. Proteome-wide XL-MS Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Developing XlinkX v2.0 search engine

5. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Solving the n-square problem
Disuccinimidyl Sulfoxide (DSSO)
Kao A., et al. Mol Cell Proteomics, 2011

6. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Solving the n-square problem
Disuccinimidyl Sulfoxide (DSSO)
Mass
Intensity
MS2 AL BL AS BS
Liu F., et al. Nat Methods, 2015

7. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Solving the n-square problem
Liu F., et al. Nat Methods, 2015

8. Proteome-wide XL-MS Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
CID-only
Liu F., et al. Nat Methods, 2015

9. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-ETD MS2 dual play
Liu F., et al. Nat Methods, 2015

10. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-ETD MS2 dual play
Ion Routing Multipole
C-Trap
Ion Trap
HPC
LPC
MP0 Q1
m/z
Full FTMS Scan
Orbitrap
m/z
1st MS2 CID Scan
m/z
2nd MS2 ETD Scan
Full OT MS scan
1st CID MS2 on MS precursor ion in OT
2nd ETD MS2 on MS precursor ion in OT
Liu F., et al. Nat Methods, 2015

11. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-only
CID-ETD MS2 dual fragmentation
Liu F., et al. Nat Methods, 2015

12. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-MS2-MS3 dual play
Liu F., et al. Manuscript in preparation

13. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-MS2-MS3 dual play
Ion Routing Multipole
C-Trap
Ion Trap
HPC
LPC
MP0 Q1
m/z
Full FTMS Scan
Orbitrap
m/z
1st MS2 CID Scan
m/z
2nd MS3 CID Scan
m/z
2nd MS3 CID Scan
m/z
2nd MS3 CID Scan
m/z
2nd MS3 CID Scan
Full OT MS scan
1st CID MS2 on MS precursor ion in OT
2nd mass difference dependent MS3 scans in IT
Liu F., et al. Manuscript in preparation

14. Solving the n-square problem
Proteome-wide XL-MS
Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Solving the n-square problem
CID-MS2-MS3 dual fragmentation
Liu F., et al. Manuscript in preparation

15. Proteome-wide XL-MS Using MS-cleavable cross-linker
Implementing combined fragmentation strategies
Developing XlinkX v2.0 search engine
Liu F., et al. Manuscript in preparation

16. Proteome-wide XL-MS application (E.coli)
Liu F., et al. Manuscript in preparation

17. Proteome-wide XL-MS application (mitochondrion)
Electron transfer chain supercomplexes (mitochondrion)
Proposed in 1955 (Chance & Williams)
Weak experimental evidence
COX7A1/2
COX5A
COX4i1
COX6B1
COX7B
CIV CI
CIII
CII
NDUFV1
NDUFA9
NDUFA5
NDUFA4
NDUFB10
NDUFA11
NDUFB4
NDUFB7
UQCRC1
UQCRC2
UQCRH
UQCR10
SDHA

18. Summary Method development of proteome-wide XL-MS
The use of MS-cleavable cross-linkers
The implement of combined fragmentation strategies
The development of XlinkX search engine (XlinkX PD nodes)
Biological applications
Intact organelles
Whole cell lysates

19. Acknowledgements Utrecht University
Prof. Albert Heck
Philip Lössl
Dr. Dirk Rijkers
Harm Post
Dr. Richard Scheltema
National Heart, Lung, and Blood Institute
Prof. Robert S. Balaban
Beverley M. Dancy
Thermo Fisher Scientific
Rosa Viner
Bernard Delanghe
Torsten Ueckert
Kai Fritzemeier